Primary Characterization of a Monoclonal Antibody … Note Primary Characterization of a Monoclonal Antibody Using Agilent HPLC-Chip Accurate-Mass LC/MS Technology The characterization

Application Note

Primary Characterization of a Monoclonal Antibody Using Agilent HPLC-Chip Accurate-Mass LC/MS Technology

The characterization of a monoclonal antibody at low nanogram levels using

an Agilent HPLC-Chip system coupled to an Agilent Accurate-Mass Q-TOF MS

instrument is described. The superior sensitivity and mass accuracy of the

HPLC-Chip LC/MS platform, combined with the powerful data processing

capabilities of Agilent MassHunter and BioConfi rm software, enabled easy and

rapid identifi cation of antibody heterogeneity and cleaved fragments. Robust,

reliable, and easy to use, Agilent nanofl ow LC/MS technology is ideally suited

for the routine analysis of biopharmaceuticals.

Introduction

Recombinant monoclonal antibodies (mAbs) represent an important class of bio-

pharmaceutical products with a wide range of diagnostic and clinical applications.

The worldwide market for monoclonal antibodies is projected to reach $26 billion by

2010–2011. These therapeutic glycoproteins are usually produced from mammalian

cell lines (hybridomas), then purifi ed, concentrated, and exchanged into an appropriate

formulation buffer prior to being sold. Although mAbs are relatively stable biomol-

ecules, a number of chemical modifi cations and degradation reactions can occur

during manufacturing, formulation, and storage, thereby necessitating reliable and

sensitive methods for the characterization of protein purity and structural integrity.

Accurate mass measurements of intact proteins, whole subunits, or domains are

useful for the rapid verifi cation of sequence composition and identifi cation of post-

translational modifi cations and sample handling artifacts.

LC/MS technology is a powerful and sensitive technique for the characterization and

identifi cation of proteins. Nanofl ow LC/MS enables faster and more sensitive protein

identifi cation while minimizing sample and solvent consumption. Agilent’s microfl uidic-

based HPLC-Chip integrates sample preparation, chromatographic separation, and

nanoelectrospray formation for effi cient, high-sensitivity nanospray LC/MS. Agilent

Accurate-Mass LC/MS TOF and Q-TOF systems deliver exceptional mass resolution,

Authors

Ravindra Gudihal

Sudha Ragalopalan

Ranjit Gulge

Andrew Gieschen

Christine Miller

Ning Tang

Keith Waddell

Agilent Technologies, Inc.,

Santa Clara, CA, USA

Application Note

2 This item is intended for Research Use Only. Not for use in diagnostic procedures.

mass accuracy, sensitivity, and data processing capabilities for

optimal MS characterization of proteins. In this note, we report

the rapid LC/MS characterization of low nanogram levels of

an intact mAb and its fragments using the Agilent HPLC-Chip

system coupled to an Accurate-Mass Q-TOF.

Experimental

Materials

L-cysteine, EDTA disodium, sodium phosphate dibasic anhy-

drous, sodium phosphate monobasic, sodium bicarbonate,

papain suspension (in 0.05 M sodium acetate), and DL-dithio-

threitol (DTT) were purchased from Sigma. PNGase and the

Tris reaction buffer pH 8.2 were obtained from Prozyme.

Deglycosylation reaction

Deglycosylation of the mAb was performed using PNGase

in 20 mM Tris-HCl buffer pH 8.2 as described by the manu-

facturer. Briefl y, 0.3 µL of 100 U/mL solution of PNGase was

added to 3 µg of antibody in 20 mM Tris-HCl buffer pH 8.2. The

mixture was incubated at 37°C for 24 hr. Samples were appro-

priately diluted in 3% acetonitrile/water with 0.1%

formic acid solution prior to LC/MS analysis.

Papain digestion

The papain was activated using freshly prepared activation

buffer (papain activation/digestion buffer: 1 mM EDTA, 50 mM

sodium phosphate buffer and 10 mM cysteine, pH 7.0) for 15

min at 37°C. Activated papain solution was then added to the

mAb in papain digestion buffer. The mAb-to-protease ratio

was 60:1(w/w). The mixture was incubated at 37°C for 2 hr.

Samples were appropriately diluted in 3% acetonitrile/water

with 0.1% formic acid solution prior to LC/MS analysis.

3

Reduction reaction

The mAb (1.5 µg) was reduced with 25 mM DTT at pH 8.6 for 1

hr at 80°C. Samples were appropriately diluted in 3% acetonitrile/

water with 0.1% formic acid solution prior to LC/MS analysis.

LC/MS Analysis

Instrumentation

The Agilent 1200 HPLC-Chip system was coupled with the

Agilent 6520 Accurate-Mass Q-TOF LC/MS platform for LC/MS

analyses (Figure 1). The HPLC-Chip is a polymer microfl uidic-

based device that integrates sample preparation (enrichment

column), analytical separation (analytical column), and nano-

electrospray formation (emitter tip). The enrichment column

helps in effi cient desalting and concentration of the sample,

reducing sample consumption. The HPLC-Chip Cube interface

enables automatic chip loading, sample and solvent delivery

to the chip, high pressure switching of fl ows, and automated

chip positioning to the MS source. The Agilent 6520 Q-TOF LC/

MS system incorporates True High-Defi nition TOF technology

to provide optimal sensitivity, mass accuracy, mass resolution,

and wide dynamic range.

LC parameters

HPLC-Chip: 5 µm, ZORBAX 300SB-C8 (300Å), 40 nL enrichment

column, and a 75 µm x 43 mm analytical column.

Flow rate: 3 µL/min from capillary pump to the enrichment

column and 300 nL/min from nano LC pump to the analytical

column.

Solvents: 0.1% formic acid in water (A); 90% acetonitrile in

water with 0.1% formic acid (B). Flush volume was set at 7 µL

for effective desalting of the mAb sample.

Sample load: 7.5-10 ng on-column for intact and deglycosy-

lated mAb while 5 ng on-column was used for reduced and

papain-generated fragments of the mAb.


www.proteomics-lab.com

Figure 1. Agilent’s HPLC-Chip technology coupled to a 6520 Accurate-Mass Q-TOF MS instrument.

HPLC-Chip

HPLC-Chip Cube 6520 Q-TOF

Application Note


Sample loading: With cap pump at 3% B.

Sample analysis: Gradient with nano pump as shown below.

MS parameters

Spectra were recorded in positive ion mode and in profi le mode

on the Agilent 6520 Accurate-Mass Q-TOF MS instrument.

Vcap: 1750 V and drying gas fl ow of 5 L/min at 325°C was

used.

Fragmentor voltage: 420 V for intact antibody/deglycosylated

form and 350 V for the fragments.

Data were acquired in standard (20,000 m/z), 1 GHz, MS only

mode, range 500-4,000 m/z (for fragments of mAb); 1,000-4,000

m/z (for intact mAb).

Data analysis

AgilentMass Hunter Qualitative Analysis software and the

MassHunter BioConfi rm software (version B.02.00) were used.

Protein and fragment masses were obtained using maximum

entropy deconvolution. Average masses for intact mAb, light

chain, heavy chain, Fab, and Fc were calculated from sequences

using the Sequence Editor feature found in the BioConfi rm

software.

4x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2706.7422

2756.7152 2611.8165

2808.7909 2566.7952

2862.7840 2523.3067

2481.2595 2919.0798

2440.5988 2977.3333

2401.2639 3038.0612 2363.1768

3101.3321 3167.3332

3308.0414

Counts vs. Mass-to-Charge (m/z)

2200 2250 2300 2350 2400 2450 2500 2550 2600 2650 2700 2750 2800 2850 2900 2950 3000 3050 3100 3150 3200 3250 3300 3350 3400

x104

0

0.5

1

1.5

2 2706.7422 2658.4255

2756.7152

2808.7909

2680.4239 2730.0683 2781.5349


2660 2680 2700 2720 2740 2760 2780 2800 2820

2658.4255

Figure 2. Mass spectrum of intact antibody with inset showing the expanded view of charge states of the antibody.

Stop time: 15 min

Time (minutes) B (%)

Initial 3

1 25

4 60

8 80

12 95

15 5



5

Results and Discussion

Analysis of Intact Antibody

Figure 2 shows the averaged mass spectrum of the intact anti-

body after elution from the HPLC-Chip. A very well distributed

series of peaks is seen corresponding to the m/z of the many

different charge state species of the intact antibody. Small

satellite peaks within each charge state are also observed

(see inset), corresponding to either adducts or modifi cations

of the mAb. To gain more insight into the possible modifi ca-

tion/adduct, deconvolution of the intact mass spectrum was

performed using a maximum entropy algorithm (Figure 3).

The deconvoluted spectrum shows three major mass peaks

at 148812.81 Da (calculated mass from sequence is 148811.95

Da; measured mass accuracy of 5.7 ppm), 147367.94 Da, and

145922.00 Da. The mass differences between the peaks are

marked in Figure 3. The observed mass difference of 1444.87

Da (calculated mass=1445.35 Da) is attributed to one unit of

the glycan (G0F) attached to the mAb. Table 1 lists some of

the commonly found N-linked glycans in the immunoglobulin.

The other observed mass difference of 2890.81 Da (calculated

mass=2890.7 Da) corresponds to two such units of G0F glycan,

indicating that the mAb sample has three species: intact mAb

with a pair of G0F glycans (148812.81 Da), mAb with one unit

Figure 3. Deconvoluted spectrum of intact antibody with inset showing the expanded view of a small amount of G1F (addition of hexose unit to G0F, see Table 1)

of the mAb.

1444.87

2890.81

G0F/G0Fx10 4

0

1

2

3

4

5

6

6.5 148812.81

145922.00

147367.94

146329.69

146816.21 147719.65

Counts vs. Deconvoluted Mass (amu)

145500 146000 146500 147000 147500 148000 148500 149000 149500 150000 150500 151000 151500

5.5

4.5

3.5

2.5

1.5

0.5

162.16 hexose unit

x104

0

1

2

3

4

5

6

7

8

148812.81

148974.97 148840.65 148916.37

148765.43

Counts vs. Deconvoluted Mass (amu) 148750 148800 148850 148900 148950

G0F/G0F

G0F/G1F

Application Note


of G0F glycan (147367.94 Da), and mAb devoid of any N-

linked glycan part (145922.00 Da). Another peak (Figure 3) at

148974.97 Da was assigned to the addition of a hexose unit to

the G0F glycan part of the antibody (observed mass increase

of 162.16 Da; calculated mass is 162.14 Da), denoted as G1F in

Figure 3 (see Table 1).

Analysis of deglycosylated antibody

To confi rm the assignment of the peak mass for the ungly-

cosylated species at mass 145922.00 Da, a deglycosylation

reaction of the mAb was performed. The mass spectrum of

the deglycosylated mAb is shown in Figure 4. The spectrum

shows well-defi ned charge state species with minimal adducts

or satellite peaks. The deconvoluted spectrum (Figure 5) has

a single molecular species at 145924.41 Da (calculated mass

Table 1. Structures of N-linked glycans commonly found in IgG molecules [2] .

Code Oligosaccharide structure Average mass Comments

G2F 1769.64

G1F 1607.49Small amount found

in this mAb

G0F 1445.35Major form found

in this mAb

G0 1299.21

Galactose

Mannose

Fucose

N-acetylglucosamine

4 x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2654.2106

2703.3116 2606.8056

2561.0999 2754.3171

2516.9783 2807.2538

2474.3228

2862.2881

2433.1203

2393.2569

2919.5091 2354.6563

2317.3069 2281.1008

2979.0715

3041.1043

3105.7939 3173.2776


1950 2000 2050 2100 2150 2200 2250 2300 2350 2400 2450 2500 2550 2600 2650 2700 2750 2800 2850 2900 2950 3000 3050 3100 3150 3200 3250 3300

Figure 4. Mass spectrum of deglycosylated antibody.



7

from sequence, and accounting for N to D conversion which

occurs during deglycation reaction, is 145923.24 Da; mass

accuracy obtained is 8 ppm), indicating removal of all glycan

moieties from the mAb. The mass obtained is also in close

agreement with the mass assignment for the unglycosylated

form (145922.00 Da) of the mAb in Figure 3.

Analysis of reduced antibody

To obtain more insight into the structure of the mAb, complete

reduction using DTT was performed. The deconvoluted spectra

of the light and heavy chains of the mAb are shown in Figure 6.

The light chain has a measured mass of 23746.5 Da (calculated

mass from sequence is 23746.63 Da; mass accuracy obtained

is -5.45 ppm) and the heavy chain has a measured mass of

50675.58 Da (calculated mass from sequence is 50675.68 Da;

mass accuracy obtained is 1.9 ppm).

No glycan attached species

4 x10

0

1

2

3

4

5

6

7

8

145924.41

146272.08


144500 145000 145500 146000 146500 147000 147500 148000 148500 149000 149500

Figure 5. Deconvoluted spectrum of deglycosylated antibody.

Application Note


Analysis of Fc and Fab fragments generated by

papain digestion

Papain is used to generate Fab and Fc fragments from a mAb

for various biotechnological/therapeutic applications. These

fragments are also useful for additional structural characteriza-

tion of a mAb. The mass spectra and deconvoluted spectra for

Fc and Fab fragments are shown in Figures 7 and 8, respec-

tively. Figure 7B further validates glycan attachment for the

intact mAb.

The Fc fragment has a measured mass of 52755.64 Da (calcu-

lated mass from sequence is 52755.62 Da; mass accuracy ob-

Figure 6. A) Deconvoluted spectrum of light chain of mAb (shown as blue bar).

B) Deconvoluted spectrum of heavy chain of mAb (shown as red bar with different sugar attachment).

5 x10

0

1

2

3

4

5

23746.50

23727.82 23762.96

Counts vs. Deonvoluted Mass (amu)

23540 23560 23580 23600 23620 23640 23660 23680 23700 23720 23740 23760 23780 23800 23820 23840 23860 23880 23900 23920 23940 23960 23980

1446.6

162.7

4 x10

0

1

2

3

4

5 50675.58

49228.96

50838.33

49435.14 51023.43 50121.16 49920.85 49009.52


48600 48800 49000 49200 49400 49600 49800 50000 50200 50400 50600 50800 51000 51200

A

B



9

tained is 0.38 ppm) and the Fab fragment has a measured mass

of 48046.09 Da (calculated mass from sequence is 48046.18

Da; mass accuracy obtained is -1.9 ppm).

Low sample consumption

In our analyses, the amount of sample consumed was in the

low nanogram level. The HPLC-Chip LC/MS method consumes

signifi cantly less sample compared to other LC methods, as

shown in Table 2, making it well suited for the analysis of small

amounts of biotherapeutics during the R&D stage or during

product release.

Figure 7. A) Mass spectrum of Fc fragment of mAb.

B) Deconvoluted mass of Fc fragment of mAb; inset shows the expanded view of small peak.

A

B

4 x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

2.2

2.3

2.4 1319.9174

1287.7236

1257.0902

1227.8910 1353.7175

1389.3139

1426.7975 1199.9867

1521.9735

1466.4584

1552.6289 1147.8771

1599.6583 1123.4876

1100.0744


1075 1100 1125 1150 1175 1200 1225 1250 1275 1300 1325 1350 1375 1400 1425 1450 1475 1500 1525 1550 1575 1600 1625 1650

1445.8

2890.93

162.47

5 x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2 52755.64

49864.71

51309.87

47485.72

50272.22


44000 44500 45000 45500 46000 46500 47000 47500 48000 48500 49000 49500 50000 50500 51000 51500 52000 52500 53000 53500 54000 54500 55000

5 x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2 52755.64

52918.11

52553.59 53081.26


52400 52600 52800 53000 53200 53400 53600 52200

Application Note


Figure 8. A) Mass spectrum of Fab fragment of mAb.

B) Deconvoluted mass of Fab fragment of mAb.

Column(dimension) Amount of sample injected onto column Reference

HPLC-Chip (43 × 0.075 mm) 5-10 ng This note and [4]

Cap LC,C8 (50 × 1 mm) ~ 4 µg [3]

Poroshell C8 (75 × 2.1 mm) ~200 ng [4]

Table 2. Sample load on different columns.

5 x10

0

0.25

0.5

0.75

1

1.25

1.5

1265.3752

1299.5473

1335.6141 1232.9565

1373.7449

1202.1551

1414.1268 1172.8591

1456.9391

1144.9538

1502.4368

1118.3572 1550.8752

1602.5404

1092.9598

1521.9702


1075 1100 1125 1150 1175 1200 1225 1250 1275 1300 1325 1350 1375 1400 1425 1450 1475 1500 1525 1550 1575 1600 1625 1650

Fab fragments

6 x10

0

0.5

1

1.5

2

48046.09

52755.52


44000 45000 46000 47000 48000 49000 50000 51000 52000 53000 54000 55000

A

B



11

Conclusions

Rapid and sensitive characterization of an intact mAb and its

fragments at low nanogram levels has been demonstrated

using Agilent’s microfl uidic HPLC-Chip/MS system coupled to

the Agilent 6520 Accurate-Mass Q-TOF MS platform. The high

sensitivity and sub-10 ppm mass accuracy of the nanofl ow LC/

MS system enabled identifi cation of mAb heterogeneity, con-

fi rmation of mAb deglycosylation, and identifi cation of Fab and

Fc fragments. This approach is ideal for the routine analysis of

small amounts of expensive and valuable biopharmaceuticals.

References

1. www.drugresearcher.com.

2. Gadgil HS, Pipes GD, Dillon TM, Treuheit MJ, Bondarenko PV. J Am Soc Mass Spectrom. 2006 Jun; 17(6):867-72.

3. Gadgil HS, Bondarenko PV, Pipes GD, Dillon TM, Banks D, Abel J, Kleemann GR, Treuheit MJ. Anal Biochem. 2006 Aug; 355(2):165-74.

4. Dayin Lin and Hongbin Liu, Protein Society 2008 Poster 236, HPLC-Chip QTOF Analysis of MAb.

Application Note

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This item is intended for Research Use Only. Not for use in diagnostic procedures. Information, descriptions, and specifi cations in this publication are subject to change without notice.

Agilent Technologies shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance, or use of this material.

© Agilent Technologies, Inc. 2008

Published in the U.S.A. December 16, 2008

5990-3445EN

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Documents

Primary Characterization of a Monoclonal Antibody … Note Primary Characterization of a Monoclonal Antibody Using Agilent HPLC-Chip Accurate-Mass LC/MS Technology The characterization